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Expression Profiles of Three Novel Sensory Guanylate Cyclases and Guanylate Cyclase-Activating Proteins in the Zebrafish Retina

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Expression Profiles of Three Novel Sensory Guanylate Cyclases and Guanylate Cyclase-Activating Proteins in the Zebrafish Retina View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1793 (2009) 1110–1114 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Expression profiles of three novel sensory guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish retina Nina Rätscho, Alexander Scholten, Karl-Wilhelm Koch ⁎ Biochemistry Group, Institute of Biology and Environmental Science, Faculty V, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany article info abstract Article history: Three membrane bound sensory guanylate cyclases are expressed in photoreceptor cells of the developing Received 30 September 2008 and adult zebrafish retina. First appearance of mRNA transcripts was detected by in situ hybridization Received in revised form 19 November 2008 techniques for all guanylate cyclases between 3 and 4 days post fertilization (dpf), but only one isoform Accepted 27 December 2008 (guanylate cyclase 3) appeared to be specifically expressed in cones of the adult retina. Transcripts of three Available online 6 January 2009 cone specific guanylate cyclase-activating proteins (zGCAP3, zGCAP4 and zGCAP7) were also detected at 3– Keywords: 4 dpf. The expression onset of the guanylate cyclases and these neuronal calcium sensor proteins mainly Guanylate cyclase overlapped. High guanylate cyclase activities in larval eye preparations and the precisely controlled GCAP coexpression of guanylate cyclases and zGCAPs coincide with the onset of visual function at 3–4 dpf. Neuronal calcium sensor protein © 2009 Elsevier B.V. All rights reserved. Retina 1. Introduction of the full-length sequences and a characterization of their molecular properties has not been reported so far. Vertebrate rod and cone photoreceptor cells respond to light by a On the molecular level excitation and adaptation in cones are less change of the membrane potential. A light-triggered enzymatic well understood than in rods. But recently, the zebrafish has attained cascade leads to the hydrolysis of the intracellular messenger increasing interest as a model organism to study cone vision. guanosine-3′,5′-cyclic monophospate (cGMP), which is synthesized Zebrafish larvae respond to visual stimuli as early as 3 days post by membrane bound guanylate cyclases [1]. While rod cells operate at fertilization (3 dpf) and electroretinogram (ERG) recordings indicate low light intensities, cone cells are sensitive at higher levels of retinal function in larvae at 4 dpf (see for example [13–16]). The ambient illumination and maintain their responsiveness at fluctuating visual system of the zebrafish contains five photoreceptor cell types: background light [2]. Adaptation to different background light rods, double cones consisting of a long and a short member (DC), intensities is mediated by an operative network of photoreceptor long single cones (LSC) and short single cones (SSC). These five cell proteins and a change of intracellular calcium concentration. Calcium types express a total of nine opsin genes (one rod opsin and eigth sensor proteins detect these changes and regulate their targets in a cone opsin genes). The absorption characteristics of the opsins in calcium dependent manner [1–6]. Among them are the recoverins and cone cells indicate that zebrafish posses colour vision similar to that the guanylate cyclase-activating proteins (GCAPs) [3–5] that belong to of human [17,18]. the family of neuronal calcium sensor proteins [6,7]. Mammalian The large variety of GCAPs in the zebrafish retina points to a GCAP1 and GCAP2 are expressed in rods and cones and regulate complex calcium-sensing regulation of membrane-bound GCs. Thus, membrane bound guanylate cyclases in a calcium-dependent way, but in the present study we investigated if the putative sensory zGCs are differ in their biochemical and physiological properties [8–10]. expressed in photoreceptors of the adult zebrafish retina and whether A larger variety of GCAPs was recently detected in retina of adult they show a temporal and spatial overlapping coexpression pattern zebrafish [11], among them are four isoforms (zGCAP3, zGCAP4, with one or more cone specific GCAPs in the larvae. zGCAP5 and zGCAP7) specifically expressed in cones [11,12]. However, almost nothing is known about guanylate cyclases in the zebrafish 2. Materials and methods retina as possible targets of zGCAPs. Only three mRNA sequences that code for putative sensory guanylate cyclases (zGCs) had been 2.1. Fish maintenance deposited in the GenBank database (gucy2f, gc2, gc3), but the cloning All experiments on zebrafish were performed in accordance with the European Communities Council Directive for animal use in science – ⁎ Corresponding author. Tel.: +49 441 798 3640; fax: +49 441 798 193640. (86/609/EEC). Fish were kept in a 14:10 h day night cycle at either E-mail address: [email protected] (K.-W. Koch). 25 °C (adult) or 28.5 °C (larvae) according to standard conditions. 0167-4889/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.12.021 N. Rätscho et al. / Biochimica et Biophysica Acta 1793 (2009) 1110–1114 1111 2.2. Preparations of larvae for whole-mount in situ hybridization sequence of gc3 was also updated (XM_690001.3), but we could not confirm this sequence information. Instead, our sequence Larvae were raised in 0.003% 1-phenyl-2-thiourea (Sigma) to information obtained by cloning strategies confirmed the sequence prevent pigmentation. If necessary chorions were removed, larvae deposited under XM_690001.2. were anesthetized by chilling on ice and immediately fixed in 4% paraformaldehyde (PFA)/1×PBS (137 mM NaCl, 2.7 mM KCl, 8 mM 2.5. In situ hybridization NaH2PO4, 2 mM KH2PO4, pH 7.4). After fixation larvae were washed in 1×PBS, dehydrated in methanol and stored at −20 °C. The 3′UTR containing regions of zGCAPs cDNA were amplified from a retinal cDNA library (library 760, Deutsches Ressourcenzentrum für 2.3. Preparation of adult retina Genomforschung GmbH, Berlin; used for zGCAP3, zGCAP4) or by cDNA first strand preparations from adult retina (zGCAP5, zGCAP7) Adult zebrafishes were anesthetized with tricaine and decapitated. using gene specific primer pairs (for zGCAP4 and zGCAP7 primer pairs Eyecups were fixed in 4% PFA/1×PBS for 20 min, cryoprotected by see [11]; for zGCAP3 and zGCAP5 see Table 1 in Supplementary bathing in 30% sucrose/1×PBS overnight at 4 °C and embedded with material). Amplified fragments were cloned into the pGEM-T-Easy liquid nitrogen in 33% Tissue Tek® (Sakura Finetech)/20% sucrose/ Vector (Promega), linearized with suitable restriction enzymes and 1×PBS and stored at −80 °C. Cryoembedded tissue was sectioned in processed for in vitro transcription of riboprobes using digoxigenin- 8–15 μm slices with a cryostat. labeled uracil (Roche). Whole-mount in situ hybridization procedure of larvae was done 2.4. In silico analyses of membrane bound retinal zebrafish guanylate as described [22]. In situ hybridization of cryosections was done cyclases and cloning of partial zGC fragments according to standard protocols [12] with slight modifications. Putative sensory guanylate cyclases were identified by two 2.6. Guanylate cyclase assay independent data base searches using the BLAST algorithm [19]:In a first step we used the partial cDNA sequences RET-GC1 (GenBank Eyes of zebrafish larvae were enucleated at 10 dpf in Ringer accession number AY050503), RET-GC2 (AY050504), and RET-GC3 solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2 and 5 mM HEPES (AY050505) published in [20] to screen the GenBank data base. pH 7.4), immediately frozen in liquid nitrogen and stored at −80 °C. The second search was done in the Danio rerio RefSeq RNA-data After thawing a buffer containing 20 mM HEPES pH 7.4, 120 mM KCl, base using the sequences for retina specific sensory guanylate 5 mM MgCl2, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl cyclases from medaka (OlGC3: GenBank accession number fluoride was added to the eyes. The suspension was homogenized and AB000899; OlGC4: AB000900; OlGC5: AB000901; OlGC-R2: used for each determination of GC activity [23]. Incubation time was AB015874; see [21]) as query. By this strategy we obtained three 30 min at 30 °C. Protein content was determined by the amido black predicted mRNA sequences for zebrafish sensory guanylate method [24]. cyclases: XM_864538.1 with a length of 3324 bp corresponds to gucy2f [20] and is the ortholog to medakafish OlGC4; XM_683407.2 3. Results (3324 bp) contains the sequence gc2 and is comparable to OlGC- R2; XM_690001.2 (3688 bp) codes for gc3 and is similar to OlGC3. 3.1. Sensory zGCs are expressed in larval and adult eyes PCR strategies were used to ensure that the predicted sequences are expressed in the zebrafish retina. For each zebrafish Zebrafish larval and adult eyes express functional GCs. In order to GC-isoform we amplified overlapping cDNA fragments using test for zGC activities we prepared whole eyes from larvae at 10 dpf, reverse transcribed RNA from adult retina as template. Primers homogenized them and measured GC activities of these preparations. designed for this PCR strategy are listed in Table 1 (Supplementary The homogenates showed rather high GC activities at 6–8 nmol cGMP material). The amplified cDNA fragments were cloned in appro- synthesized/min and mg protein. Similar results were obtained with priate cloning vectors and verified by DNA sequencing. For gucy2f adult eye preparations. Recently, we were able to show that guanylate and gc2 the confirmed sequences were recently updated cyclases in membranes of adult zebrafish retina can be activated by (NM_131864.2, gucy2f and NM_001109695.1, gc2). The predicted recombinant zGCAP4 [25]. Fig. 1. Comparison of amino acid sequences of zGC3 with human ROS-GC2 (A) and human GC-A (B). Analysis was performed with the program PDOTPLOT, version 3.13 (Dr.
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